Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

PTP-MEG2 is an intracellular protein tyrosine phosphatase with a putative lipid-binding domain at the N-terminus. The present study reports expression, purification, and characterization of the full-length form of the enzyme plus a truncated form containing the catalytic domain alone. Full-length PTP-MEG2 was expressed with an adenovirus system and purified from cytosolic extracts of human 293 cells infected with the recombinant adenovirus. The purification scheme included chromatographic separation of cytosolic extracts on fast flow Q-Sepharose, heparin-agarose, l-histidyldiazobenzylphosphonic acid agarose, and hydroxylapatite. The enrichment of PTP-MEG2 from the cytosol was about 120-fold. The truncated form of PTP-MEG2 was expressed in E. coli cells as a non-fusion protein and purified by using a chromatographic procedure similar to that used for the full-length enzyme. The purified full-length and truncated enzymes showed single polypeptide bands on SDS-polyacrylamide gel electrophoresis under reducing conditions and behaved as monomers on gel exclusion chromatography. With para-nitrophenylphosphate and phosphotyrosine as substrates, both forms of the enzyme exhibited classical Michaelis-Menten kinetics. Their responses to pH, ionic strength, metal ions, and protein phosphatase inhibitors are similar to those observed with other characterized tyrosine phosphatases. Compared with full-length PTP-MEG2, the truncated DeltaPTP-MEG2 displayed significantly higher V(max) and lower K(m) values, suggesting that the N-terminal putative lipid-binding domain may have an inhibitory role. The full-length and truncated forms of PTP-MEG2 were also expressed as GST fusion proteins in E. coli cells and purified to near homogeneity through affinity columns. However, the specific phosphatase activities of the GST fusion proteins were 10-25-fold below those obtained with the correspondent non-fusion proteins.
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PMID:Purification and characterization of protein tyrosine phosphatase PTP-MEG2. 1211 18

A novel protein phosphatase in Arabidopsis thaliana was identified by database searching. This protein, designated AtPTPKIS1, contains a protein tyrosine phosphatase (PTP) catalytic domain and a kinase interaction sequence (KIS) domain. It is predicted to interact with plant SNF1-related kinases (SnRKs), representing central regulators of metabolic and stress responses. AtPTPKIS1 has close homologues in other plant species, both dicots and monocots, but is not found in other kingdoms. The tomato homologue of AtPTPKIS1 was expressed as a recombinant protein and shown to hydrolyse a generic phosphatase substrate, and phosphotyrosine residues in synthetic peptides. The KIS domain of AtPTPKIS1 was shown to interact with the plant SnRK AKIN11 both in vivo in the yeast two-hybrid system, and in vitro in a GST-fusion 'pull down' assay. The genomes of Arabidopsis and other plants contain further predicted proteins related to AtPTPKIS1, which could also interact with SnRKs and act in novel regulatory and signalling pathways.
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PMID:A novel higher plant protein tyrosine phosphatase interacts with SNF1-related protein kinases via a KIS (kinase interaction sequence) domain. 1214 29

Signal transducer and activator of transcription (STAT) proteins are both tyrosine- and serine-phosphorylated, mediating signal transduction and gene regulation. Following gene regulation, STAT activity in the nucleus is then terminated by a nuclear protein phosphatase(s), which remains unidentified. Using novel antibody arrays to screen the Stat1-specific protein phosphatase(s), we identified a SHP-2-Stat1 interaction in the A431 cell nucleus. SHP-2 and Stat1 nuclear localization and their association in response to either epidermal growth factor or interferon-gamma (IFNgamma) were confirmed by immunofluorescent staining and affinity precipitation assays. The SHP-2 C-terminal region containing protein-tyrosine phosphatase activity interacted with the C-terminal SH2 transcriptional activation domain of Stat1. In SHP-2-/- mouse fibroblast cells, Stat1 phosphorylation at both the tyrosine residue Tyr(701) and the serine residue Ser(727) by IFNgamma was enhanced and prolonged. Consistently, purified GST-SHP-2 dephosphorylated Stat1 at both tyrosine and serine residues when immunoprecipitated phospho-Stat1 or a peptide corresponding to the sequence surrounding Tyr(P)(701) or Ser(P)(727) of Stat1 was used as the substrate. Overexpression of SHP-2 in 293T cells inhibited IFNgamma-dependent Stat1 phosphorylation and suppressed Stat1-dependent induction of luciferase activity. Our findings demonstrate that SHP-2 is a dual-specificity protein phosphatase involved in Stat1 dephosphorylation at both tyrosine and serine residues and plays an important role in modulating STAT function in gene regulation.
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PMID:SHP-2 is a dual-specificity phosphatase involved in Stat1 dephosphorylation at both tyrosine and serine residues in nuclei. 1227 Sep 32

Numerous reports have revealed that the tumor suppressor BRCA1 may play an important role in DNA damage repair. BRCA1 is expressed and phosphorylated during cell cycle progression and after DNA damage. BRCA1 is hypophosphorylated in G0-G1 and probably during mitosis as well. Kinases known to phosphorylate BRCA1 include cyclin-dependent kinase 2, as well as ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related kinase (ATR), which function in G2 checkpoint control. However, protein phosphatases responsible for dephosphorylation of BRCA1 had yet to be identified. hCds1, which acts downstream of ATM, also phosphorylates a BRCA1 fragment containing amino acids 759-1064 [BRCA1 fragment 4 (BF4)]. We have used a GST-BF4 protein phosphorylated by hCds1 [glutathione S-transferase (GST)-BF4-P] as a substrate to identify potential phosphatases responsible for BRCA1 dephosphorylation. Data presented here show that both recombinant protein phosphatase 1 alpha (PP1alpha) catalytic subunit and endogenous PP1alpha dephosphorylate GST-BF4-P. Inhibitor 2 abolishes this activity. Overexpression of PP1alpha partially inhibits hyperphosphorylation of BRCA1 after ionizing radiation, indicating that PP1alpha dephosphorylates BRCA1 in vivo. BRCA1 and PP1alpha reciprocally coimmunoprecipitate, and a glutathione S-transferase pull-down assay shows that PP1alpha catalytic subunit associates directly with the BF4 region of BRCA1. In addition, BRCA1 inhibits PP1alpha activity. Therefore, BRCA1 is both a substrate and a regulator of PP1alpha. The interaction between BRCA1 and PP1alpha thus may play a role in DNA damage repair and cell cycle progression.
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PMID:Regulation of BRCA1 phosphorylation by interaction with protein phosphatase 1alpha. 1243 14

Reversible phosphorylation is recognized to be a major mechanism for the control of intracellular events in eukaryotic cells. From a human fetal brain cDNA library, we isolated a cDNA clone encoding a novel dual specificity protein phosphatase, which showed 88% identity with previously reported mouse LMW-DSP3 at the amino acid level. The deduced protein had a single dual-specificity phosphatase catalytic domain, and lacked a cdc25 homology domain. LMW-DSP3 was expressed in the heart, lung, liver, and pancreas, and the expression level in the pancreas was highest. The LMW-DSP3 gene was located in human chromosome 2q32, and consisted of five exons spanning 21kb of human genomic DNA. LMW-DSP3 fused to GST showed phosphatase activity towards p-nitrophenyl phosphate which was optimal at pH 7.0 and 40 degrees C, and the activity was enhanced by Ca(2+) and Mn(2+). The phosphatase activity of LMW-DSP3 was inhibited by orthovanate. LMW-DSP3 showed phosphatase activity toward oligopeptides containing pSer/Thr and pTyr, indicating that LMW-DSP3 is a protein phosphatase with dual substrate specificity.
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PMID:Molecular cloning and characterization of a novel human protein phosphatase, LMW-DSP3. 1247 73

The catalytic subunit of type 1 serine/threonine protein phosphatase (PP1c) was shown to bind trithorax (TRX) in the yeast two-hybrid system. Interaction between PP1c and TRX was confirmed in vivo by co-immunoprecipitation from Drosophila extracts. An amino-terminal fragment of TRX, containing a putative PP1c-binding motif, was shown to be sufficient for binding to PP1c by in vitro glutathione S-transferase pull-down assays using recombinant protein and fly extracts expressing epitope tagged PP1c. Disruption of the PP1c-binding motif abolished binding, indicating that this motif is necessary for interaction with PP1. On polytene chromosomes, PP1c is found at many discrete bands, which are widely distributed along the chromosomes. Many of the sites that stain strongly for PP1c correspond to sites of TRX, consistent with a physical association of PP1c with chromatin-bound TRX. Homeotic transformations of haltere to wing in flies mutant for trx are dominantly suppressed by PP1c mutants, indicating that PP1c not only binds TRX, but is a physiologically relevant regulator of TRX function in vivo.
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PMID:Trithorax interacts with type 1 serine/threonine protein phosphatase in Drosophila. 1252 22

PNUTS, Phosphatase 1 NUclear Targeting Subunit, is a recently described protein that targets protein phosphatase 1 (PP1) to the nucleus. In the present study, we characterized the biochemical properties of PNUTS. A variety of truncation and site-directed mutants of PNUTS was prepared and expressed either as glutathione S-transferase fusion proteins in Escherichia coli or as FLAG-tagged proteins in 293T cells. A 50-amino acid domain in the center of PNUTS mediated both high affinity PP1 binding and inhibition of PP1 activity. The PP1-binding domain is related to a motif found in several other PP1-binding proteins but is distinct in that Trp replaces Phe. Mutation of the Trp residue essentially abolished the ability of PNUTS to bind to and inhibit PP1. The central PP1-binding domain of PNUTS was an effective substrate for protein kinase A in vitro, and phosphorylation substantially reduced the ability of PNUTS to bind to PP1 in vitro and following stimulation of protein kinase A in intact cells. In vitro RNA binding experiments showed that a C-terminal region including several RGG motifs and a novel repeat domain rich in His and Gly interacted with mRNA and single-stranded DNA. PNUTS exhibited selective binding for poly(A) and poly(G) compared with poly(U) or poly(C) ribonucleotide homopolymers, with specificity being mediated by distinct regions within the domain rich in His and Gly and the domain containing the RGG motifs. Finally, a PNUTS-PP1 complex was isolated from mammalian cell lysates using RNA-conjugated beads. Together, these studies support a role for PNUTS in protein kinase A-regulated targeting of PP1 to specific RNA-associated complexes in the nucleus.
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PMID:PNUTS, a protein phosphatase 1 (PP1) nuclear targeting subunit. Characterization of its PP1- and RNA-binding domains and regulation by phosphorylation. 1257 61

Dual-specificity protein phosphatases (DSPs), a new family of protein tyrosine phosphatases (PTPs), are characterized by the ability to dephosphorylate both phospho-tyrosyl and phospho-seryl/threonyl residues. It has been known that most of the enzymes play important roles in the regulation of mitogenic signal transduction and control the cell cycle in response to extracellular stimuli. In this study, a novel human DSP gene named Dual-specificity Phosphatase18 (DUSP18) was isolated by large-scale sequencing analysis of a human fetal brain cDNA library. DUSP18 is localized at Chromosome 22 q12.1. Its cDNA is 2450 base pairs in length, encoding a 188-amino acid polypeptide in which a DSP motif but not a CH2 domain is included. RT-PCR revealed that the DUSP18 was widely expressed in different tissues. GST-DUSP18 fusion protein showed distinctive phosphatase activity toward p-nitrophenyl phosphate (pNPP), as well as oligopeptides containing pThr and pTyr, indicating that DUSP18 is a protein phosphatase with dual substrate specificity. The optimal condition for the reaction was pH 6.0 and 55 degrees C. Addition of Mn(2+) ions was able to enhance the enzyme activity while the activity was strongly inhibited by iodoaretic acid. Mutations in selected sites showed the importance of Asp-73, Cys-104, Arg-110 and Ser-111 in phosphatase activity of DUSP18.
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PMID:Molecular cloning and characterization of a novel dual-specificity phosphatase18 gene from human fetal brain. 1259 17

G-protein-coupled receptor kinase 2 (GRK2) is known to specifically phosphorylate the agonist-bound forms of G-protein-coupled receptors (GPCRs). This strict specificity is due at least partly to activation of GRK2 by agonist-bound GPCRs, in which basic residues in intracellular regions adjacent to transmembrane segments are thought to be involved. Tubulin was found to be phosphorylated by GRK2, but it remains unknown if tubulin can also serve as both a substrate and an activator for GRK2. Purified tubulin, phosphorylated by GRK2, was subjected to biochemical analysis, and the phosphorylation sites in beta-tubulin were determined to be Thr409 and Ser420. In addition, the Ser444 in beta III-tubulin was also indicated to be phosphorylated by GRK2. The phosphorylation sites in tubulin for GRK2 reside in the C-terminal domain of beta-tubulin, which is on the outer surface of microtubules. Pretreatment of tubulin with protein phosphatase type-2A (PP2A) resulted in a twofold increase in the phosphorylation of tubulin by GRK2. These results suggest that tubulin is phosphorylated in situ probably by GRK2 and that the phosphorylation may affect the interaction of microtubules with microtubule-associated proteins. A GST fusion protein of a C-terminal region of beta I-tubulin (393-445 residues), containing 19 acidic residues but only one basic residue, was found to be a good substrate for GRK2, like full-length beta-tubulin. These results, together with the finding that GRK2 may phosphorylate synuclein and phosducin in their acidic domains, indicate that some proteins with very acidic regions but without basic activation domains could serve as substrates for GRK2.
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PMID:Identification of sites of phosphorylation by G-protein-coupled receptor kinase 2 in beta-tubulin. 1263 Dec 74

The p21-activated kinase(Pak) 2 undergoes rapid autophosphorylation/activation in neutrophils stimulated with a variety of chemoattractants (e.g., fMLP). Phosphorylation within the activation loop (Thr(402)) and inhibitory domain (Ser(141)) is known to increase the activity of Pak in vitro, whereas phosphorylation within the Nck (Ser(20)) and Pak-interacting guanine nucleotide exchange factor (Ser(192) and Ser(197)) binding sites blocks the interactions of Pak 2 with these proteins. A panel of phosphospecific Abs was used to investigate the phosphorylation of Pak 2 in neutrophils at these sites. Pak 2 underwent rapid (< or =15 s) phosphorylation at Ser(20), Ser(192/197), and Thr(402) in neutrophils stimulated with fMLP. Phosphorylation at Ser(192/197) and Thr(402) were highly transient events, whereas that at Ser(20) was more persistent. In contrast, Pak 2 was constitutively phosphorylated at Ser(141) in unstimulated neutrophils and phosphorylation at this site was less sensitive to cell stimulation than at other residues. Studies with selective inhibitors suggested that a variety of phosphatases might be involved in the rapid dephosphorylation of Pak 2 at Thr(402) in stimulated neutrophils. This was consistent with biochemical studies which showed that the activation loop of GST-Pak 3, which is homologous to that in Pak 2, was a substrate for protein phosphatase 1, 2A, and a Mg(2+)/Mn(2+)-dependent phosphatase(s) which exhibited properties different from those of the conventional isoforms of protein phosphatase 2C. The data indicate that Pak 2 undergoes a complex pattern of phosphorylation in neutrophils and that dephosphorylation at certain sites may involve multiple protein phosphatases that exhibit distinct modes of regulation.
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PMID:p21-activated kinase 2 in neutrophils can be regulated by phosphorylation at multiple sites and by a variety of protein phosphatases. 1450 Jun 79


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